4072 The Journal of Experimental Biology 212, 4072-4078 Published by The Company of Biologists 2009 doi:10.1242/jeb.032466 Moths are not silent, but whisper ultrasonic courtship songs R. Nakano1,*,†, T. Takanashi2, T. Fujii1, N. Skals3, A. Surlykke4 and Y. Ishikawa1 1Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan, 2Department of Forest Entomology, Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan, 3Vedbendvænget 6, DK-5800 Nyborg, Denmark and 4Center for Sound Communication, Institute of Biology, University of Southern Denmark, DK-5230, Odense M, Denmark *Present address: Laboratory for Biolinguistics, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan †Author for correspondence ([email protected]) Accepted 1 September 2009 SUMMARY Ultrasonic hearing is widespread among moths, but very few moth species have been reported to produce ultrasounds for sexual communication. In those that do, the signals are intense and thus well matched for long distance communication. By contrast, males of the Asian corn borer moth (Crambidae) were recently shown to whisper extremely low-intensity ultrasonic courtship songs close to females. Since low sound levels will prevent eavesdropping by predators, parasites and conspecific rivals, we predicted low intensity ultrasound communication to be widespread among moths. Here we tested 13 species of moths including members of the Noctuidae, Arctiidae, Geometridae and Crambidae. Males of nine species, 70%, produced broadband ultrasound close to females. Peak frequencies ranged from 38 to above 100kHz. All sounds were of low intensity, 43–76dBSPL at 1cm [64±10dBpeSPL (mean ± s.d.), N9 species]. These quiet and/or hyper-frequency ultrasounds are audible to nearby mates, but inaudible to unintended receivers. Although largely unknown because it is so inconspicuous, acoustic communication using low intensity ultrasound appears to be widespread among hearing moths. Thus, acoustic communication may be the norm rather than the exception. Supplementary material available online at http://jeb.biologists.org/cgi/content/full/212/24/4072/DC1 Key words: acoustic communication, courtship behavior, low sound level, moths, ultrasound. INTRODUCTION not been verified in most cases, but one effect of the male songs Most moths have tympanal ears sensitive to ultrasound. The tuning seems to be to prompt their mates to accept mating, in some cases of moth hearing to bat calls (Fullard, 1998) as well as the in conjunction with chemical signals as in the pyralid, Galleria deterioration of moth hearing in bat-free areas (Surlykke et al., 1998) mellonella (Spangler, 1985). Given that many arctiid moths produce indicates that ears probably evolved in response to selective ultrasonic clicks to counteract bats (Miller and Surlykke, 2001), pressures imposed by foraging bats (Hoy and Robert, 1996; Miller sexual communication via ultrasounds is likely to be a secondary and Surlykke, 2001; Waters, 2003). The location and morphology use of the ability to produce ultrasound (Endler and Basolo, 1998). of moth ears vary across moth superfamilies, indicating an We recently added the Asian corn borer moth, Ostrinia furnacalis independent development in each group after the divergence of moth (Crambidae), to the list of moths that produce courtship songs. O. superfamilies (Hoy and Robert, 1996; Greenfield, 2002; Yack, furnacalis shows mating behavior typical of the majority of moths. 2004). First, males of O. furnacalis are attracted toward a sex pheromone- Subsequent to the development of ears, a relatively small number releasing female. After landing near the female, the male approaches of moth species developed sound producing organs, and used them her with his wings fanning (<2cm) and subsequently attempts to either for defense against bats or rival males (Spangler, 1988; mate by bending his abdominal tip toward hers. Male moths were Conner, 1999; Greenfield, 2002; Waters, 2003). For instance, both found to produce ultrasonic courtship songs of extremely low sexes of tiger moths (Arctiidae) emit jamming or warning ultrasonic intensity (46dBpeSPL at 1cm) during these copulation attempts clicks in response to echolocation calls of bats (Surlykke and Miller, (Nakano et al., 2006; Nakano et al., 2008). Since the use of quiet 1985; Barber and Conner, 2007; Ratcliffe and Nydam, 2008). In sound would be advantageous in avoiding eavesdropping by the lesser wax moth, Achroia grisella (Pyralidae), males produce predacious bats and rival males, this result prompted us to predict loud ultrasonic clicks to attract female mates (Spangler, 1988). The that whispering of ultrasonic courtship songs is widespread among ultrasounds used by these species are characterized by high sound moths (Nakano et al., 2008; Nakano et al., 2009). In the present pressure levels, with the peak equivalent sound pressure level study, we tested our prediction by examining 13 tympanate moth (peSPL) at a distance of 1cm ranging from 76 to 125dB. species, belonging to diverse taxonomic groups, for the production Production of ‘courtship’ sounds, the sounds produced after pair of quiet ultrasonic courtship songs. formation and before mating, has only rarely been reported in moths, e.g. in some arctiids (Conner, 1987; Krasnoff and Yager, 1988; MATERIALS AND METHODS Sanderford and Conner, 1990; Simmons and Conner, 1996; Insects Sanderford et al., 1998) and pyralids (Spangler, 1985; Trematerra We investigated the emission of courtship ultrasound in 13 and Pavan, 1995). The acoustic communication between sexes has tympanate moth species belonging to four families (three THE JOURNAL OF EXPERIMENTAL BIOLOGY Moth ultrasonic courtship songs 4073 superfamilies): five species of Noctuidae (Noctuoidea), the cotton Amyna natalis (Heller and Achmann, 1993), Hecatesia exultans bollworm Helicoverpa armigera Hübner, the cabbage armyworm (Alcock and Bailey, 1995), Heliothis zea (Kay, 1969), Amphipyra Mamestra brassicae Linnaeus, the common cutworm Spodoptera perflua (Lapshin and Vorontsov, 2000), Syntomeida epilais litura Fabricius, Herminia tarsicrinalis Knoch and Diomea cremata (Sanderford and Conner, 1995), Cycnia tenera (Barber and Conner, Butler; three species of Arctiidae (Noctuoidea), the tiger moth 2006), Arctia caja (Surlykke and Miller, 1985), Euchaetes egle Spilosoma punctarium Stoll, the fall webworm Hyphantria cunea (Simmons and Conner, 1996), Phragmatobia fuliginosa (Surlykke Drury and Eilema japonica Leech; one species of Geometridae and Miller, 1985), Pyrrharctia isabella (Krasnoff and Yager, 1988), (Geometroidea), the Japanese giant looper Ascotis selenaria cretacea Euchaetes bolteri (Simmons and Conner, 1996), Empyreuma affinis Butler; and four species of Crambidae (Pyraloidea), the rice stem (Sanderford et al., 1998), Bena bicolorana (Skals and Surlykke, borer Chilo suppressalis Walker, the mulberry pyralid Glyphodes 1999), Pseudoips prasinana (Skals and Surlykke, 1999), Corcyra pyloalis Walker, Palpita nigropunctalis Bremer and Spoladea cephalonica (Spangler, 1987), Achroia grisella (Spangler, 1984), recurvalis Fabricius (see supplemental TableS1 for details). Symmoracma minoralis (Heller and Krahe, 1994), and 36 tiger moths Larvae of H. armigera, M. brassicae, S. litura, H. tarsicrinalis, described by Barber and Conner (Barber and Conner, 2006). E. japonica and A. selenaria were reared on a commercial artificial Differences in the sound pressure levels emitted by the two groups diet for silkworms, SilkmateTM 2M (Nosan Corp., Yokohama, were analyzed with a generalized linear model (GLM) using the Japan), and the other larvae were reared on their host plants under software package R, version 2.7.0. conditions of 24°C, 50–70% relative humidity, and a 16h:8h light:dark cycle. Pupae and/or adults were separated by sex, based Behavioral experiments on the genital morphology of the terminal abdominal segment, and To verify that the sounds were used for sexual communication in S. maintained under the same environmental conditions as for rearing. litura, we examined how mating was affected by deafening females. Emerged female and male moths were transferred to 430ml plastic For the operation, 0- or 1-day-old virgin females were anesthetized cups with a supply of water until used. with CO2, and the tympanic membranes on both sides of the metathorax were punctured using a fine insect pin under a Ultrasound recordings stereomicroscope. The sham operation involved puncturing another A single unmated male and five to ten 1- to 4-day-old virgin females part of the metathorax. Behavioral experiments were conducted in were introduced into a cubic mesh cage (18cmϫ18cmϫ18cm) the last 2h of the scotophase 1day after the operation. A single 1- placed in a soundproof box (40cmϫ40cmϫ70cm) with one side day-old intact unmated male was introduced into a cubic mesh cage open, and maintained for >1h before a recording. The activities of (25cmϫ25cmϫ25cm), which housed 5–10 deafened, sham-operated, the insects were observed under a dim red light (0.2lux). Sound or intact females. Multiple females were housed in the cage so that recordings were made when the moths showed high mating activity, always, at least one female would be releasing sex pheromone during i.e. during the last 3h of the scotophase. The exception was C. the experiment. In response to the sex pheromone released by the suppressalis, which showed high mating activity during the early females, the male usually readily flew